Labelled oligonucleotides synthesized on solid-supports

ABSTRACT

Methods and compositions to label oligonucleotides and analogs directly on a solid-support having the structure  
                 
 
where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P 1  is an acid cleavable protecting group are provided. The labelled solid-support is reacted in a cyclical fashion to synthesize a labelled oligonucleotide on a solid-support in the 5′ to 3′ direction, having the structure:  
                 
Labelled oligonucleotides are also synthesized by reacting: (i) a label reagent bearing functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on solid support with nucleophilic functionality, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling reaction may be conducted at label sites including the 5′ terminus, the 3′ terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending application Ser. No.10/371,985, filed Feb. 20, 2003, which is a continuation of applicationSer. No. 10/010,717, filed Nov. 7, 2001, now U.S. Pat. No. 6,525,183,which is a continuation of application Ser. No. 09/813,378, filed Mar.20, 2001, now U.S. Pat. No. 6,316,610, which is a continuation ofapplication Ser. No. 09/256,340, filed Feb. 22, 1999, now U.S. Pat. No.6,255,476, which are all incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of nucleic acid chemistry,and particularly to methods and compositions for labellingoligonucleotides on solid-supports. Label reagents includehybridization-stabilizing moieties, fluorescent dyes, fluorescencequenchers, energy-transfer dye sets, chemiluminescent dyes, metalloporphyrins, amino acids, proteins, peptides, enzymes, and affinityligands.

REFERENCES

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BACKGROUND

Non-isotopically labelled oligonucleotides are essential components inmany important molecular biology applications, such as PCRamplification, DNA sequencing, antisense transcriptional andtranslational control of gene expression, genetic analysis, and DNAprobe-based diagnostic testing (Keller, 1993; Kricka, 1992).Fluorescence detection of fluorescent dye-labelled oligonucleotides isthe basis for nucleic acid sequence detection assays such as Taqman™(Livak, 1996), Molecular Beacons (Tyagi, 1996), genetic linkage mapping(Dib, 1996), and oligonucleotide-ligation assay (Grossman, 1994).

Two general methods for labeling synthetic oligonucleotides have beenestablished. In a first method, referred to herein as the “two-stepsolution labelling method”, a nucleophilic functionality, e.g. a primaryaliphatic amine, is introduced at a labelling attachment site on anoligonucleotide, e.g. a 5′ terminus. After automated, solid-supportsynthesis is complete, the oligonucleotide is cleaved from the supportand all protecting groups are removed. The nucleophile-oligonucleotideis reacted with an excess of a label reagent containing an electrophilicmoiety, e.g. isothiocyanate or activated ester, e.g.N-hydroxysuccinimide (NHS), under homogeneous solution conditions(Hermanson, 1996; Andrus, 1995).

In a second alternative method, referred to herein as the “directlabeling method”, a label is directly incorporated into theoligonucleotide during or prior to synthesis (Mullah, 1998; Nelson,1992). The direct labelling method is preferred because it (i) does notrequire a post-synthesis reaction step, thereby simplifying thesynthesis of labelled polynucleotides; and (ii) avoids the problemsassociated with the low reaction yield (<60%) typically encountered withthe two-step solution labelling method, namely: (a) purification of thelabeled oligonucleotide from excess label; (b) purification of thelabeled oligonucleotide from unlabeled oligonucleotide; (c) high costsdue to the low product yield and laborious analytical and purificationprocedures, and; (d) irreversible capping of the nucleophilicfunctionality during synthesis.

Certain fluorescent dyes and other labels have been functionalized asphosphoramidite reagents for 5′ labelling (Theisen, 1992). However, somelabels, e.g., digoxigenin, rhodamine dyes, and cyanine dyes, are toounstable to survive the harsh conditions and reagents used in reagentpreparation and oligonucleotide synthesis, cleavage and deprotection.Thus, whenever such labels are used in current solid phase synthesisprotocols, they must be attached using the less preferred two-stepsolution labelling method.

Therefore it is desirable to provide methods and reagents to labeloligonucleotides and analogs directly on a solid-support upon which theyare synthesized, under conditions which are rapid, economical, andcompatible with chemically-labile functionality.

SUMMARY

The present invention is directed toward novel methods and compositionsfor synthesis of labelled oligonucleotides on solid-supports.

In a first aspect, the invention comprises a method for synthesis oflabelled oligonucleotides on a labelled solid-support having thestructure

where S is a solid-support, A is a cleavable linker, X is a moiety withthree or more attachment sites, L is a label, Y is a nucleophile, i.e.O, NH, NR or S, and P₁ is an acid cleavable protecting group. Thelabelled solid-support is reacted in a cyclical fashion with reagentsto: (1) remove P₁ from Y, (2) couple Y with the 5′ position of a5′-phosphoramidite, 3′ protected nucleoside, (3) cap any unreacted siteson the support, e.g. Y, if necessary, and (4) oxidize any phosphitelinkages. The four steps are repeated until the entire labelledoligonucleotide is synthesized.

After synthesis is complete, protecting groups on the internucleotidephosphates and nucleobases of the labelled oligonucleotide may beremoved by deprotection while the oligonucleotide remains on thesolid-support. Alternatively, after synthesis is complete, the labelledoligonucleotide may be cleaved from the solid-support and thendeprotected.

In a second aspect, the invention comprises a nucleoside bound to asolid-support having the structure

where S, A, X, L, and Y are defined as before, R is a phosphateprotecting group or phosphotriester substituent; B is a nucleobase; P₂is an exocyclic nitrogen protecting group; and P₃ is an acid-labileprotecting group.

In a third aspect, the invention comprises an oligonucleotide bound to asolid-support having the structure

where the variable substituents are defined as before, and n is aninteger preferably from about 0 to 100.

In a fourth aspect, the invention comprises a method for synthesizing alabelled oligonucleotide by reacting: (i) a label reagent bearingfunctionality that can be converted into an electrophile, e.g.carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid,(ii) an oligonucleotide on a solid support with a nucleophilicfunctionality, e.g. alcohol, amine, or thiol, and (iii) a couplingreagent, whereby an ester, amide, thioester, sulfonamide, sulfonate,phosphonate, phosphoramidate, phosphorothioate, or phosphate bond isformed. The labelling method may be conducted on an oligonucleotide atlabel sites including the 5′ terminus, the 3′ terminus, a nucleobase, aninternucleotide linkage, a sugar, amino, sulfide, hydroxyl, andcarboxyl. The labelling reaction may be conducted on an oligonucleotidecomprising one or more DNA, RNA, PNA and nucleic acid analog monomerunits. The nucleic acid analogs may be nucleobase, sugar, and/orinternucleotide analogs.

The labelled oligonucleotide may be synthesized either in the 5′ to 3′direction, or in the 3′ to 5′ direction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Oligonucleotide attached to labelled-support at 5′ terminus

FIG. 2 Synthetic route to support-linker-label reagents 1 to 6.

FIG. 3 Fluorescent dye labels: FAM, TET, HEX, JOE, NED, VIC

FIG. 4 Fluorescent dye labels: dJON, dR139, JODA and energy-transferdonor FAM

FIG. 5 Fluorescence quencher labels: TAMRA, ROX, DABCYL, DABSYL, NTB

FIG. 6. Minor groove binder labels: MGB1, CDPI monomer, CDPI₃

FIG. 7 Coupling of TAMRA labelling reagent with HBTU coupling reagent to5′-aminohexyl oligonucleotide on solid-support

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

The terms “nucleic acid”, “polynucleotide” or “oligonucleotide” meanpolymers of nucleotide monomers or analogs thereof, including double andsingle stranded deoxyribonucleotides, ribonucleotides, α-anomeric formsthereof, and the like. The oligonucleotide may comprise one or more DNA,RNA, and nucleic acid analog monomer units. The monomers are linked byinternucleotide linkages, including phosphodiester bonds or phosphateanalogs thereof, and associated counterions, e.g., H⁺, NH₄ ⁺, Na⁺.Oligonucleotides typically range in size from a few monomeric units,e.g. 5-40, to several thousands of monomeric units. Whenever anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′ to 3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, unless otherwise noted.

“Nucleoside” refers to a compound consisting of a purine, deazapurine,or pyrimidine nucleobase, e.g., adenine, guanine, cytosine, uracil,thymine, deazaadenine, deazaguanosine, and the like, linked to a pentoseat the 1′-position. When the nucleoside base is purine or 7-deazapurine,the pentose is attached to the nucleobase at the 9-position of thepurine or deazapurine, and when the nucleobase is pyrimidine, thepentose is attached to the nucleobase at the 1-position of thepyrimidine.

“Nucleotide” refers to a phosphate ester of a nucleoside, e.g., atriphosphate ester, wherein the most common site of esterification isthe hydroxyl group attached to the C-5 position of the pentose.

The term “Watson/Crick base-pairing” refers to a pattern of specificpairs of nucleotides, and analogs thereof, that bind together throughsequence-specific hydrogen-bonds, e.g. A pairs with T and U, and G pairswith C.

The term “analog” refers to analogs of nucleic acids made from monomericnucleotide analog units, and possessing some of the qualities andproperties associated with nucleic acids. Nucleic acid analogs may havemodified nucleobase moieties, modified sugar moieties, and/or modifiedinternucleotide linkages (Englisch, 1991). A preferred class of nucleicacid analogs in which the sugar and internucleotide moieties have beenreplaced with an 2-aminoethylglycine amide backbone polymer is peptidenucleic acids PNA (Nielsen, 1991).

“Attachment site” refers to a site to which a linker is attached.

“Linker” refers to one or more atoms comprising a chain connecting anoligonucleotide to a label or a solid-support.

“Chimera” as used herein refers to an oligonucleotide including one ormore nucleotide and one or more nucleotide analog units.

“Lower alkyl”, “lower alkylene” and “lower substituted alkylene” refersto straight-chain, branched, or cyclic groups consisting of 1-12 carbonatoms.

“Label” refers to a moiety covalently attached to an oligonucleotide. Apreferred class of labels provides a signal for detection, e.g.fluorescence, chemiluminescence, and electrochemical luminescence(Kricka, 1992). Another preferred class of labels serve to enhance,stabilize, or influence hybridization, e.g. intercalators, minor-groovebinders, and cross-linking functional groups (Blackburn, 1996).Detection labels include, but are not limited to, fluorescent dyes, suchas fluorescein and rhodamine derivatives, cyanine dyes (Kubista, 1997),chemiluminescent dyes (Bronstein, 1990; Bronstein, 1994) andenergy-transfer dyes (Clegg, 1992; Cardullo, 1988). Yet anotherpreferred class of labels serve to effect the separation orimmobilization of a molecule by specific or non-specific capture means(Andrus, 1995).

“Detection” refers to detecting, observing, or measuring anoligonucleotide on the basis of the properties of a label.

“Coupling reagents” include any reagent, activator, or additive that canform an ester, amide, thioester, sulfonamide, sulfonate, phosphonate,phosphoramidate, phosphorothioate, or phosphate bond between thenucleophile oligonucleotide on solid-support and the label.

II. Labelled-Supports

In one aspect of the present invention comprises supports for thesynthesis of labelled oligonucleotides. The labelled supports accordingto this aspect of the present invention have the structure:

where S refers generally to a solid-support material useful foroligonucleotide synthesis, A is a cleavable linker, X is a moiety withthree or more attachment sites, L is a label, Y is a nucleophile, i.e.O, NH, NR or S, and P₁ is an acid cleavable protecting group.

The solid-supports provide an insoluble media for sequential attachmentof monomer units. A significant advantage of heterogeneous synthesismethods is that excess reagents in the liquid phase may be easilyremoved by filtration, thereby eliminating the need for purificationsteps between each reaction or each cycle. The characteristics of thesolid-support, such as pore size, cross-link content, swelling, particlesize, and surface area, should be optimized to allow for efficientdiffusion of reagents in order to give rapid kinetics and high-yieldreactions. Preferred support materials include high cross-link,non-swelling polystyrene (Andrus, 1993), controlled-pore-glass(Caruthers, 1984), silica, silica gel, polyacrylamide, magnetic beads(Stamm, 1995), polyacrylate, hydroxyethylmethacrylate, polyamide,polyethylene, polyethyleneoxy, and copolymers or grafts of such.Physical configurations of solid-supports include small particles,beads, membranes, frits, non-porous surfaces, slides, plates,micromachined chips, alkanethiol-gold layers, addressable arrays,vectors, plasmids, or polynucleotide-immobilizing media (Fodor, 1995).In some embodiments, it may be desirable to create an array ofphysically separate synthesis regions on the support with, for example,wells, raised regions, dimples, pins, trenches, rods, inner or outerwalls of cylinders, and the like.

The cleavable linker A may be comprised of any functionality such as:(i) esters and other base-labile linkers that are cleaved by basicreagents, (ii) silyl ethers that are cleaved by nucleophiles such asfluoride, or (iii) disulfide groups and other groups that are cleavedunder oxidation/reduction conditions with reagents such asdithiothreitol (DTT). The bonds that are cleaved in above examples oflinkers A are shown by the arrows below:

The moiety X may be any group comprised of attachment sites forattachment of the solid-support through linker A, a label L, and theoligonucleotide.

Labels L are any group or moiety covalently attached to theoligonucleotide. Labels may be comprised of hybridization-stabilizingmoieties, (e.g. minor-groove binders, intercalators, and cross-linkingagents), fluorescent dyes, fluorescence quenchers, energy-transfer dyesets, chemiluminescent dyes, amino acids, proteins, peptides, enzymes,and affinity ligands.

Y is a group nucleophilic relative to carbon, e.g. O, NH, NR, and S andattaches X to the oligonucleotide. Y has an acid-cleavable protectinggroup, P₁, which may be DMT, MMT, trityl, substituted trityl, pixyl, ortrialkylsilyl. The protecting group P₁ is removed to commenceoligonucleotide synthesis from the nucleophile Y.

III. Synthesis of Labelled-Supports

The solid-supports S are derivatized with reactive functionality towhich is attached a linker unit, A-X-Y. Preferably the reactivefunctionality is a primary amino group with a preferable loading of5-100 NH₂ per gram. A linker unit, or spacer, A-X-Y, is then covalentlyattached to the reactive functionality of the solid-support. A secondattachment site on A-X-Y attaches to labels. A third attachment site onA-X-Y allows synthesis of the oligonucleotide chain. Typically Acontains an ester group which is cleavable under basic conditions, e.g.ammonium hydroxide, to allow separation of the solid-support from theoligonucleotide and any labels attached to the oligonucleotide.

The linker unit A-X-Y may be attached to the solid-support as: (i) oneunit with a label L, or (ii) as a unit without a label L, where thelabel L is then attached to the linker X by the reactions:

FIG. 2 shows the exemplary route to a labelled-support (ii) where thelinker 1 (X-Y-P₁) is converted to 2 (A-X-Y-P₁) and attached toaminomethyl, highly-cross linked polystyrene 3. The resulting product 4is deprotected to 5. A pre-activated label, e.g.TAMRA-NHS(N-hydroxysuccinimide ester of 5-carboxy tetramethylrhodamine)is covalently attached to 5 to yield the labelled-support 6, ready foroligonucleotide synthesis.

A preferred group of labelled solid-supports is embodied in thestructure 6 (FIG. 2 and below)

where S is high cross-link polystyrene or controlled-pore glass,

-   A is-   X is-   L is minor-groove binder, cyanines, fluorescent dyes, or    energy-transfer dye sets, Y is oxygen, and P₁ is DMT.

The asymmetric carbon in X of 6 leads to diastereomer isomers oflabelled oligonucleotides. These particular isomers have the advantageof not resolving into separate peaks during analysis by HPLC orcapillary electrophoresis. Other diastereomeric linkers for attachinglabels to oligonucleotides may show diastereomeric resolution,exemplified by double peaks in analysis (Mullah, 1998; Woo, 1996). Wherelabelled oligonucleotides prepared from 6 are used in primer extensionexperiments such as DNA sequencing (Lee, 1997), DNA fragment analysis(Grossman, 1994) and PCR (Livak, 1996), the fragments and amplificationproducts are similarly advantaged by not separating into diastereomericpopulations which can hinder data analysis.

IV. Synthesis of Labelled Oligonucleotides on Labelled-Support in the 5′to 3′ Direction

Generally, the methods and compositions of the present invention utilizethe phosphoramidite synthesis method, preferred because of its efficientand rapid coupling and the stability of the starting nucleoside monomers(Caruthers, 1983; Beaucage, 1983; Beaucage, 1992). The phosphoramiditemethod entails cyclical addition of nucleotide monomer units to anoligonucleotide chain growing on a solid-support, most commonly in the3′ to 5′ direction in which the 3′ terminus nucleoside is attached tothe solid-support at the beginning of synthesis. The method is usuallypracticed using automated, commercially available synthesizers (PEBiosystems, Caruthers, 1984). The 5′ to 3′ direction embodiment of thepresent invention cyclically adds a 5′-phosphoramidite, 3′ protectednucleoside monomer (Wagner, 1997) having the structure 7:

where, R is a protecting group or substituent, e.g. cyanoethyl, methyl,lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl; R₁and R₂ are amine substituents, e.g. isopropyl, morpholino, methyl,ethyl, lower alkyl, cycloalkyl, and aryl; P₂ is an exocyclic nitrogenprotecting group such as benzoyl, isobutyryl, acetyl, phenoxyacetyl,aryloxyacetyl, dimethylformamidine, dialkylformamidine, anddialkylacetamidine; and P₃ is an acid-labile protecting group such asDMT, MMT, pixyl, trityl, and trialkylsilyl.

An oligonucleotide is synthesized with the 5′ terminus attached to thesolid-support and a free, unattached 3′ terminus (FIG. 1).

The following briefly describes the steps of a synthesis cycle, in thepresent invention, using the phosphoramidite method. First, a solidsupport, e.g. 6, is treated with a protic acid, e.g., trichloroaceticacid or dichloroacetic acid, to remove an acid labile protecting group,e.g., DMT, freeing a nucleophile, e.g. hydroxyl, for a subsequentcoupling reaction. An activated intermediate is then formed bysimultaneously adding a 5′-phosphoramidite, 3′ protected nucleosidemonomer 7 and a weak acid, e.g. tetrazole or the like, to the reactionvessel on the synthesizer, i.e. synthesis column. Nucleoside addition istypically complete within 30 to 300 s, preferably about 90 s. Next, acapping step may be performed which terminates any oligonucleotidechains that did not undergo nucleoside addition by acylation of the 3′hydroxyl. Capping is preferably done with acetic anhydride and1-methylimidazole, but other acylating agents may be used. Theinternucleotide linkage is then converted from the phosphite to the morestable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor to give the structure:

Alternatively, the internucleotide phosphite can be oxidized to aninternucleotide analog such as phosphorothioate or phosphoramidate.After oxidation, the next 3′ hydroxyl protecting group, e.g. DMT, isremoved with the protic acid, e.g., trichloroacetic acid ordichloroacetic acid, and the cycle is repeated until chain elongation iscomplete (FIG. 1).

V. Post-Synthesis Labelling of Oligonucleotides on Solid-Support

Labels may be attached at various attachment sites on oligonucleotidesand nucleic acid analogs, including: (i) a terminus, e.g. 5′ and/or 3′termini of probes, (ii) an internucleotide linkage, (iii) a sugar, or(iv) a nucleobase. Labels are most conveniently and efficientlyintroduced at the 5′ terminus, the labelling site which leastdestabilizes hybridization and least interferes with 3′ primer-extensionreactions (Kricka, 1992; Hermanson, 1996). A preferred 5′ linker reagentis a protected-amino, phosphoramidite with the structure 8:

where R is an oxygen protecting group or substituent, e.g. cyanoethyl,methyl, lower alkyl, substituted alkyl, phenyl, aryl, or substitutedaryl; and R₁ and R₂ are amino substituents, e.g. isopropyl, morpholino,methyl, ethyl, lower alkyl, cycloalkyl, or aryl. Coupling of the abovereagent with a 5′-hydroxyl group of a support-bound oligonucleotide anda weak acid activator, e.g. tetrazole, yields the monomethoxytrityl(MMT) protected oligonucleotide. After phosphite oxidation, the MMTgroup can be removed from the amine group with the same acid reagent,e.g. TCA or DCA, to unveil the reactive primary amine nucleophile forcoupling with a label reagent. The hexyl linker can easily be replacedby other inert linkers of shorter, e.g. ethyl, or longer length, e.g.dodecyl, also including aryl groups and other functionality.Alternatively, thiol or hydroxyl nucleophiles can be introduced at the5′ terminus or other sites on an oligonucleotide bound to asolid-support. Preferred thiol-protected phosphoramidite reagents are 9and 10:

Such reagents may be similarly coupled to the 5′ hydroxyl group,phosphite oxidized, and thiol protecting group removed to give areactive thiol nucleophile. Resulting 5′-linkednucleophile-oligonucleotides bound to a solid-support may be representedas:

The labelling reaction is conducted between: (i) an oligonucleotidebound to a solid-support where the oligonucleotide has reactivenucleophilic functionality, e.g. HO, HNR, H₂N, or HS, (ii) a label withCO₂H (carboxyl), SO₃H (sulfonate), RPO₃H (phosphonate), or OPO₃H(phosphate) functionality, (iii) a coupling reagent, and (iv) a solventor mixture of solvents. The reaction may be conducted at a temperaturebetween 0-100° C. and preferably at ambient temperature of about 20° C.

Preferred coupling reagents include HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),TBTU (2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH (N,N′,N″,N′″-tetramethyluronium2-fluoro-hexafluorophosphate), BOP(benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), HOBt(1-hydroxybenzotriazole), N-hydroxysuccinimide, MSNT(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl sulfonylhalides, e.g. triisopropylbenzenesulfonyl chloride.

Prior or separate activation (“pre-activation”) of a label functionalityis thereby not necessary to the practice of the present invention. Forexample, the present invention does not require prior conversion of acarboxyl group to an NHS ester for reaction with anucleophile-oligonucleotide (FIG. 7).

VI. Labels

A label L may be any moiety covalently attached to an oligonucleotide ornucleic acid analog.

A preferred class of labels are detection labels, which may provide asignal for detection of the labelled oligonucleotide by fluorescence,chemiluminescence, and electrochemical luminescence (Kricka, 1992).Fluorescent dyes useful for labelling oligonucleotides includefluoresceins (Menchen, 1993), rhodamines (Bergot, 1994), cyanines(Kubista, 1997), and metal porphyrin complexes (Stanton, 1988).

Examples of fluorescein dyes include 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE),2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein(NED), 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and(JODA) (FIGS. 3 and 4). The 5-carboxyl, and other regio-isomers, mayalso have useful detection properties. Fluorescein and rhodamine dyeswith 1,4-dichloro substituents are especially preferred.

Another preferred class of labels include quencher moieties. Theemission spectra of a quencher moiety overlaps with a proximalintramolecular or intermolecular fluorescent dye such that thefluorescence of the fluorescent dye is substantially diminished, orquenched, by fluorescence resonance energy transfer (FRET).Oligonucleotides which are intramolecularly labelled with bothfluorescent dye and quencher moieties are useful in nucleic acidhybridization assays, e.g. the “Taqman™” exonuclease-cleavage PCR assay(Livak, 1998; Livak, 1996).

Particularly preferred quenchers include but are not limited to (i)rhodamine dyes selected from the group consisting oftetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine(ROX), and (ii) DABSYL, DABCYL, cyanine dyes including nitrothiazoleblue (NTB), anthraquinone, malachite green, nitrothiazole, andnitroimidazole compounds and the like (FIG. 5).

Fluorescein (left) and rhodamine (right) derivatives of the presentinvention may bear the general structure and numbering system below,where X is a linker, and may be substituted at one or more of thenumbered positions.

Cyanine labels may have the structure

where R₁ or R₂ is H, lower alkyl, lower alkylene, lower substitutedalkylene, phenyl, or aryl; Z is CR₁R₂, S, O, NH, or N—R₁; R₃ is nitro,halo, sulfonate, hydroxy, amino, lower alkyl, or trihalomethyl, andn=0-2 (Kubista, 1997). The attachment site X for labelling ofoligonucleotides may be at R₁, R₂, or R₃.

Energy-transfer dyes are a preferred class of oligonucleotide labels. Anenergy-transfer dye label includes a donor dye linked to an acceptor dye(Lee, 1998). Light, e.g. from a laser, at a first wavelength is absorbedby a donor dye, e.g. FAM. The donor dye emits excitation energy absorbedby the acceptor dye. The acceptor dye fluoresces at a second wavelength,with an emission maximum about 100 nm greater than the absorbancemaximum of the donor dye.

The donor dye and acceptor dye moieties of an energy-transfer label maybe attached by a linker such as

linking the 4′ or 5′ positions of the donor dye, e.g. FAM, and a 5- or6-carboxyl group of the acceptor dye.

Metal porphyrin complexes are also a preferred class of oligonucleotidelabels (Stanton, 1988). One example is aluminum phthalocyaninetetrasulfonate, structure shown below:

Another preferred class of labels comprise chemiluminescent compounds.Particularly preferred are 1,2-dioxetane chemiluminescent moieties(Bronstein, 1994; Bronstein, 1990) having the structure

where R₁ is hydrogen or halogen; R₂ is phosphate, galactoside,glucoside, glucuronide, trialkylsilyloxy, acyloxy, or hydrogen; R₃ ismethyl, ethyl, and lower alkyl, and X is a linker to an oligonucleotide.Affinity ligands include biotin, 2,4-dinitrophenyl, digoxigenin,cholesterol, polyethyleneoxy, and peptides.

Another preferred class of labels, referred to herein ashybridization-stabilizing moieties, include but are not limited to minorgroove binders, intercalators, polycations, such as poly-lysine andspermine, and cross-linking functional groups. Hybridization-stabilizingmoieties may increase the stability of base-pairing, i.e. affinity, orthe rate of hybridization, exemplified by high thermal meltingtemperatures, T_(m), of the duplex. Hybridization-stabilizing moietiesserve to increase the specificity of base-pairing, exemplified by largedifferences in T_(m) between perfectly complementary oligonucleotide andtarget sequences and where the resulting duplex contains one or moremismatches of Watson/Crick base-pairing (Blackburn, 1996). Preferredminor groove binders include Hoechst 33258, CDPI₁₋₃, MGB1, netropsin,and distamycin (FIG. 6). An example of a minor groove binder is CDPI₃(Kutyavin, 1996; Lukhtanov, 1995) having the structure

where X is a linker or attachment site for labeling of oligonucleotides.When labelled to oligonucleotides, minor groove binders may increase theaffinity and specificity of hybridization to some or substantially mosttarget sequences (Blackburn, 1996, p. 337-46).VII. Nucleic Acid Analogs

Oligonucleotides may contain various nucleic acid analogs bearingmodifications to the nucleobase, sugar, and/or internucleotide moieties.

Preferred nucleobase analog modifications include but are not limited toC-5-alkyl pyrimidines, 2-thiopyrimidine, 2,6-diaminopurine, C-5-propynepyrimidine, phenoxazine (Flanagan, 1999), 7-deazapurine, isocytidine,pseudo-isocytidine, isoguanosine, 4(3H)-pyrimidone, hypoxanthine,8-oxopurines and universal base (Meyer, 1994).

Preferred sugar analog modifications in one or more of the nucleosidesinclude but are not limited to 2′- or 3′-modifications where the 2′- or3′-position may be hydrogen, hydroxy, methoxy, ethoxy, allyloxy,isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido,amino, alkylamino, fluoro, chloro and bromo.

Other preferred sugar analog modifications include 4′-α-anomericnucleotides, 1′-α-anomeric nucleotides, 2′-branchinggroup-ribonucleotides, and 2′-O-branching group-ribonucleotides. Thestructure below illustrates several preferred 2′-sugar modifications.

Preferred internucleotide analogs between one or more nucleotidesinclude but are not limited to: (i) substitution of oxygen in theinternucleotide linkage by sulfur, carbon, or nitrogen, and (ii)sulfate, carboxylate, and amide internucleotide phosphodiester linkages.Other preferred internucleotide analogs include; 2-aminoethylglycine(PNA), 2′-5′-linkage, inverted 3′-3′ linkage, inverted 5′-5′ linkage,phosphorothioate, phosphorodithioate, methyl phosphonate, non-bridgingN-substituted phosphoramidate, alkylated phosphotriester branchedstructure, and 3′-N-phosphoramidate.

An especially preferred analog of the present invention is thepeptide-nucleic acid oligomer, PNA, in which the naturalphosphodiester-deoxyribose backbone has been replaced byN-(2-aminoethyl)-glycine, a peptide-like unit (Nielsen, 1991). PNAoligomers are capable of base-pairing with complementary sequences inthe clamp-specific portion of the probe by Watson/Crick base-pairing.PNA and PNA/DNA chimera can be synthesized using conventional methods oncommercially available, automated synthesizers, with commerciallyavailable reagents (Dueholm, 1994; Vinayak, 1997; Van der Laan, 1997).

VIII. Cleaving and Deprotecting Labelled Oligos from Supports

After synthesis, the oligonucleotide may be left on the solid-support,or it may be removed or separated from the solid-support by a cleavagereaction. It is desirable to leave the oligonucleotide on thesolid-support because some nucleic acid hybridization assays employoligonucleotides covalently bound to a solid-support in configurationssuch as non-porous surfaces, planar-surface arrays, enclosed orencapsulated particles, or loose particles suspended in aqueous media.

Oligonucleotides may be cleaved from the support using a base, e.g.,ammonium hydroxide, tert-butyl amine, methylamine, ethylamine, potassiumcarbonate, or sodium hydroxide. A preferred solution for cleavage anddeprotection is a mixture of methanol: tert-butyl amine:water (1:1:2,v:v:v) (Woo, 1993). The cleavage solution also removes phosphateprotecting groups, e.g., cyanoethyl, and protecting groups on theexocyclic amines of the nucleobases upon heating theoligonucleotide-containing solution at an elevated temperature, e.g.,55-85° C. for a period of 1 to 16 hours.

Another preferred cleavage method is reductive or oxidative cleavage ofa disulfide linker A where A is—(CH₂)_(n)—S—S—(CH₂)_(n)—where n is 1 to 12. A preferred cleavage reagent is dithiothreitol(DTT).

Another preferred cleavage method is fluoride ion cleavage of a silylether linkage A where A is

and where R is lower alkyl of 1 to 20 carbon atoms. Preferred cleavingreagents include tetrabutyl ammonium fluoride and hydrogenfluoride/triethylamine.

IX. EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of thepresent invention and not to in any way limit its scope.

Example 1 Synthesis of Polystyrene Support-Linker-TAMRA 6

A solution of diglycolic anhydride (64 mg, 0.55 mmol) in CH₂Cl₂ (5 ml)was added to a mixture of Et₃N (67 mg, 0.66 mmol),4-dimethylaminopyridine (34 mg, 0.28 mmol) and compound 1 (400 mg, 0.55mmol) in CH₂Cl₂ (15 ml) at 0° C. under argon atmosphere (FIG. 2). Afterthe addition was complete (10 min), the ice bath was removed and thereaction mixture was stirred at room temperature for 1 h. The reactionmixture was diluted with CH₂Cl₂ (30 ml) and washed with 5% aqueouscitric acid (1×50 ml) and saturated brine (2×50 ml). The organic layerwas dried (MgSO₄) and evaporated to give a foam. The product waspurified by column chromatography on silica gel eluting with aCHCl₃-EtOH gradient (2-10% EtOH). Appropriate fractions were combinedand evaporated to give compound 2 as a colorless foam (260 mg, 56%). ¹HNMR (CDCl₃) d: 1.20 (m, 2H), 1.39 (m, 2H), 1.58 (m, 2H), 2.18 (t, J=7.5Hz, 2H), 2.90-3.25 (m, 4H), 3.80 (s, 6H), 3.86 (s, 4H), 4.00-4.40 (m,6H), 4.85 (unresolved t, 1H), 5.92 (d, J=7.2 Hz, 1H), 6.75 (d, J=8.1 Hz,4H), 7.20-7.40 (m, 13H), 7.52 (d, J=7.2 Hz, 2H), 7.69 (d, J=7.2 Hz, 2H).

Highly cross-linked polystyrene 3 (1000 Å, 10 μmol/g amine loading, 1 g,10 μmol), was treated with compound 2 (17 mg, 20 μmol), HOBt (3 mg, 20μmol), HBTU (8 mg, 20 μmol), and diisopropylethylamine (8 mg, 60 μmol)in DMF (10 ml) on a wrist-action shaker for 4 h at room temperature togive 4. The support was washed with DMF (3×10 ml), CH₃CN (2×10 ml) andCH₂Cl₂ (1×10 ml) and dried under high vacuum overnight. A ninhydrinassay showed 0.5 μmol/g amine left on the support. The support wascapped with acetic anhydride/lutidine in THF (10% solution, 5 ml) and1-methylimidazole in THF (16% solution, 5 mL) for 2 h at roomtemperature. Support 4 was washed with CH₃CN (3×10 ml) and CH₂Cl₂ (1×10ml). Trityl cation assay gave 9.2 μmol/g loading of compound 2 on thepolystyrene support. Support 4 was treated with 20% piperidine in DMF(3×10 ml, 10 min each wash) to remove the Fmoc protecting group to givesupport 5, which was washed with DMF (3×10 ml), CH₃CN (2×10 ml) andCH₂Cl₂ (1×10 ml) and, dried under vacuum overnight. Support 5 (1 g, 9.2mmol) was treated with TAMRA-NHS ester (15 mg, 28.5 μmol) and Et₃N (8.6mg, 85 μmol) in DMF (10 mL) at room temperature for 36 h on a shaker togive support 6 (L=TAMRA). The support was washed with DMF (3×10 ml),CH₃CN (2×10 ml) and CH₂Cl₂ (1×10 ml) and dried under high vacuum for 24h. Ninhydrin test indicated less than 0.5 μmol/g amine left on thesupport. The support was capped with acetic anhydride/lutidine in THF(10% solution, 5 ml) and 1-methylimidazole in THF (16% solution, 5 ml)for 1 h and then washed with CH₃CN (3×10 ml), CH₂Cl₂ (2×10 ml) and driedunder high vacuum for 24 h. The trityl cation assay showed a finalloading of 8.8 μmol/g for polystyrene support-linker-TAMRA 6.

Example 2 Synthesis of FAM-M13-21 Primer on Labelled-Support 6

Synthesis of the FAM-M13-21 Primer: 5′ FAM-TGTAAAACGACGGCCAGT 3′ SEQ.ID. NO. 1was conducted on the ABI 394 DNA/RNA synthesizer (Perkin-Elmer Co.) at0.2 μmole scale with FAM-labelled, polystyrene support 6, (FIG. 2,L=6-carboxyfluorescein FAM, S=polystyrene) The standard 0.2 μmol CEcycle was modified by increasing the coupling time from 25 s to 90 s forcoupling of all 5′-phosphoramidite, 3′-DMT phosphoramidite nucleosides 7(Glen Research). After synthesis in the 5′ to 3′ direction was complete,FAM-M13-21 primer was cleaved and deprotected in MeOH:t-BuNH₂:H₂O(1:1:2) at 65° C. for 3 h. The primer was analyzed by conventionalmeans, i.e. anion-exchange HPLC and used in DNA sequencing.

Example 3 Labelling of Amino-207av 18mer Oligonucleotide onSolid-Support with TAMRA-CO₇H

Synthesis of 5′ TCACAGTCTGATCTCGAT 3′ was conducted on the ABI 394DNA/RNA synthesizer (Perkin-Elmer Co.) at 0.2 μmole scale withunlabelled polystyrene support in the 3′ to 5′ direction with 5′-DMT,3′-phosphoramidite nucleosides (A^(bz), G^(dmf), C^(bz), T). Theamino-linker phosphoramidite reagent 8 (Glen Research) was coupled asthe final monomer and detritylated with 3% trichloroacetic acid inCH₂Cl₂. The synthesis column, bearing the 5′-amino-207avoligonucleotide, was removed from the synthesizer. Two luer-tipped 1 mlsyringes were mounted on each end of the synthesis column. One syringewas filled with 10.7 mg (25 μmole) of TAMRA-CO₂H in 500 μl drydimethylformamide (DMF). The second syringe was filled with 9.25 mg (25μmole) HBTU and 9 μl (50 μmole) diisopropylethylamine in 250 μl of 1:1,DMF:CH₃CN. The coupling reagents in the syringes were passed through thecolumn by sequentially depressing each plunger. After thorough mixingfor about one minute, the assembly was left to stand for about 15minutes for the coupling reaction to proceed. The reagents werewithdrawn into one syringe and discarded. The synthesis column waswashed with 5 ml 1:1, DMF:CH₃CN, 5 ml CH₃CN, and treated with 1 mlMeOH:t-BuNH₂:H₂O (1:1:2) to cleave TAMRA-N-207av: 5′TAMRA-N-TCACAGTCTGATCTCGAT 3′ SEQ. ID. NO. 2from the support. The supernatant containing TAMRA-N-207av was heatedand deprotected at 65° C. for 3 h to remove all protecting groups.TAMRA-N-207av was analyzed by reverse-phase HPLC and MALDI-TOF massspectroscopy which confirmed homogeneous purity and identity.

Example 4 Labelling of Thiol-Oligonucleotide with CDPI3-CO₂H

Synthesis of 5′ TCACAGTCTGATCTCGAT 3′ is conducted on the ABI 394DNA/RNA synthesizer (Perkin-Elmer Co.) at 0.2 μmole scale withunlabelled polystyrene support in the 3′ to 5′ direction with 5′-DMT,3′-phosphoramidite nucleosides (A^(bz), G^(dmf), C^(bz), T). Thethiol-linker phosphoramidite reagent 10 is coupled as the final monomerand detritylated with silver nitrate in DMF. The synthesis column,bearing the 5′-thiol-207av oligonucleotide, is removed from thesynthesizer. Two luer-tipped 1 ml syringes are mounted on each end ofthe synthesis column. One syringe is filled with 15 mg (25 μmole) ofCDPI₃-CO₂H (FIG. 6, CDPI₃, X=OH) in 500 μl dry dimethylformamide (DMF).The second syringe is filled with 9.25 mg (25 μmole) HATU and 9 μl (50μmole) diisopropylethylamine in 250 μl of 1:1, DMF:CH₃CN. The couplingreagents in the syringes are passed through the column by sequentiallydepressing each plunger. After thorough mixing for about one minute, theassembly is left to stand for about 15 minutes for the coupling reactionto proceed. The reagents are withdrawn into one syringe and discarded.The synthesis column is washed with 5 ml 1:1, DMF:CH₃CN, 5 ml CH₃CN, andtreated with 1 ml MeOH:t-BuNH₂:H₂O (1:1:2) to cleave CDPI₃-S-207av: 5′CDPI₃-S-TCACAGTCTGATCTCGAT 3′ SEQ. ID. NO. 3from the support. The supernatant containing CDPI₃-207av is heated anddeprotected at 65° C. for 3 h to remove all protecting groups.CDPI₃-S-207av is analyzed by reverse-phase HPLC to confirm homogeneouspurity. Analysis by MALDI-TOF mass spectroscopy confirms identity.

Example 5 Labelling of 5′ Amino-SG1 Oligonucleotide on Solid-Supportwith NTB-CO₂H

Synthesis of 5′ ATGCCCTCCCCCATGCCATCCTGCGT 3′ was conducted on the ABI394 DNA/RNA synthesizer (Perkin-Elmer Co.) at 0.2 μmole scale withunlabelled polystyrene support in the 3′ to 5′ direction with 5′-DMT,3′-phosphoramidite nucleosides (A^(bz), G^(dmf), C^(bz), T). Theamino-linker phosphoramidite reagent 8 (Glen Research) was coupled asthe final monomer and detritylated with 3% trichloroacetic acid inCH₂Cl₂. The synthesis column, bearing the 5′-amino-SG1 oligonucleotide,was removed from the synthesizer. Two luer-tipped 1 ml syringes weremounted on each end of the synthesis column. One syringe was filled with11 mg (25 μmole) of NTB-CO₂H in 500 μl dry dimethylformamide (DMF). Thesecond syringe was filled with 9.25 mg (25 μmole) HBTU and 9 μl (50μmole) diisopropylethylamine in 250 μl of 1:1, DMF:CH₃CN. The couplingreagents in the syringes were passed through the column by sequentiallydepressing each plunger. After thorough mixing for about one minute, theassembly was left to stand for about 15 minutes for the couplingreaction to proceed. The reagents were withdrawn into one syringe anddiscarded. The synthesis column was washed with 5 ml 1:1, DMF:CH₃CN, 5ml CH₃CN, and treated with 1 ml MeOH:t-BuNH₂:H₂O (1:1:2) to cleaveNTB-N-SG1: SEQ. ID. NO. 4 5′ NTB-N-ATGCCCTCCCCCATGCCATCCTGCGT 3′from the support. The supernatant containing NTB-SG1 was heated anddeprotected at 65° C. for 3 h to remove all protecting groups. NTB-SG1was analyzed by reverse-phase HPLC to confirm homogeneous purity.MALDI-TOF mass spectroscopy gave molecular mass of 8390.27 whichconfirmed identity.

Example 6 Labelling of SG1 on Solid-Support with NTB-Phosphate and MSNT

Synthesis of 5′ ATGCCCTCCCCCATGCCATCCTGCGT 3′ was conducted on the ABI394 DNA/RNA synthesizer (Perkin-Elmer Co.) at 0.2 μmole scale withunlabelled polystyrene support in the 3′ to 5′ direction with 5′-DMT,3′-phosphoramidite nucleosides (A^(bz), G^(dmf), C^(bz), T). The 5′terminus hydroxyl group was detritylated with 3% trichloroacetic acid inCH₂Cl₂. The synthesis column, bearing the 5′-hydroxyl-SG1oligonucleotide, was removed from the synthesizer. Two luer-tipped 1 mlsyringes were mounted on each end of the synthesis column. One syringewas filled with 12 mg (25 μmole) of NTB-phosphate in 500 μl dry DMF. Thesecond syringe was filled with 7.5 mg (25 μmole) MSNT and 9 μl (50μmole) diisopropylethylamine in 250 μl DMF. The coupling reagents in thesyringes were passed through the column by sequentially depressing eachplunger. After thorough mixing for about one minute, the assembly isleft to stand for about 60 minutes for the coupling reaction to proceed.The reagents were withdrawn into one syringe and discarded. Thesynthesis column is washed with 5 ml 1:1, DMF:CH₃CN, 5 ml CH₃CN, andtreated with 1 ml MeOH:t-BuNH₂:H₂O (1:1:2) to cleave NTB-P-SG1: SEQ. ID.NO. 5 5′ NTB-P-ATGCCCTCCCCCATGCCATCCTGCGT 3′from the support. The supernatant containing NTB-P-SG1 is heated anddeprotected at 65° C. for 3 h to remove all protecting groups. NTB-P-SG1is analyzed by reverse-phase HPLC and MALDI-TOF mass spectroscopy toconfirm homogeneous purity and identity.

Example 7 Labelling of PNA on Solid-Support with MGB1

Automated synthesis of PNA and PNA/DNA chimera was performed using anABI Model 394 DNA/RNA synthesizer or 433A peptide synthesizer(Perkin-Elmer Co.) according to the general procedures described in thesynthesizer manufacturer's Users Manual, as well as Egholm, 1993.

PNA were synthesized at 2-5 μmole scale on MBHA (methylbenzhydrylamine)linker, high-loaded polystyrene support, and with standard synthesistechniques and nucleobase (A^(bz), C^(bz), G^(ibu), T) and primary amino(MMT, Fmoc or Boc) protecting groups, essentially as previously reported(Dueholm, 1994). A 3 ml reaction vessel is used at the 5 μmole scalewith a total reaction volume of 440 μl.

PNA were prepared with a carboxy-terminal lysine on MBHA solid support,by preloading with t-Boc-lys(Fmoc). PNA with carboxy-terminal amideswere synthesized either directly on an MBHA support or on a MBHA supportpre-loaded with the t-Boc T PNA monomer. All resins were loaded to 0.1to 0.25 mmole/g. A spacer O, 2-(2-aminoethoxy) acetic acid, can becoupled as the Fmoc-amino protected synthon. One or more spacer O unitsact as a flexible, non-base pairing, hinge region in PNA sequences.

The support used for PNA/DNA chimera synthesis is a non-swelling,high-cross linked polystyrene bead with a hydroxymethylbenzoic acidlinker (Vinayak, 1997). PNA monomers for chimera synthesis use the MMTgroup for primary amino protection. In the first step, the monomer, HATUand DIPEA, each dissolved in DMF/CH₃CN, 1/1, are delivered concurrentlyto the reaction cartridge. After 16 min, capping reagents are delivered.To minimize the tendency of the primary amino function of PNA to migrateor cyclize, the amino terminus is acetylated after removal of the finalMMT group. Reagents have been described to link DNA and PNA moieties,and other procedures for chimera synthesis, cleavage, deprotection, andpurification (Van der Laan, 1997). In this approach, the chimera can bemade continuously in a single cartridge and on a single synthesizer.

PNA oligomer H₂N-TCCTCCTT (1 μmole) on solid-support was synthesized bythe above procedures. The PNA on polystyrene support was reacted with amixture of MGB1-CO₂H (5 mg, 10 μmole, FIG. 6, Gong, 1997), HATU (10μmole), 5 μl DIEA and 100 μl DMF and allowed to stand for 1 hour at roomtemperature. The support was then washed with DMF and CH₂Cl₂, cleavedwith TFMSA (trifluoromethanesulfonic acid) at room temperature for 1hour, and precipitated in ether to give MGB1-PNA: MGB1-TCCTCCTT SEQ. ID.NO. 6MGB1-PNA was analyzed by reverse-phase HPLC and MALDI-TOF massspectroscopy which confirmed homogeneous purity and identity.

Example 8 Labelling of PNA on Solid-Support with CDPI₃

By the same procedures and reagents as Example 9, CDPI₃ was attached tothe PNA H₂N-TCCTCCTT by three consecutive couplings of Fmoc-CDPI (FIG.6, Lukhtanov, 1995) to give CDPI₃-labelled PNA. The CDPI monomer unit,1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate, protected with Fmoc(5 mg, 0.012 mmole) was dissolved in 100 μl NMP and activated by 0.95equivalents HATU (0.2M in DMF) and 2 equivalents DIEA (0.4 m in DMF).After one hour at room temperature, the activated Fmoc-CDPI solution wasadded to the support bound PNA and allowed to couple for another hour atroom temperature. The support was washed following the coupling with 20ml DMF. The Fmoc was removed by treatment of the resin support with 1:4piperidine:DMF for 10 minutes at room temperature. This coupling anddeprotection cycle was repeated two additional times for a total of 3manual couplings. The support was then washed with DMF and CH₂Cl₂,followed by cleavage with TFMSA (trifluoromethanesulfonic acid) at roomtemperature for 1 hour, followed by ether precipitation of the crudeCDPI₃-PNA: CDPI₃-TCCTCCTT SEQ. ID. NO. 7CDPI₃-PNA was analyzed by reverse-phase HPLC and MALDI-TOF massspectroscopy which confirmed homogeneous purity and identity

Example 9 Labelling of Tagman Self-Quenching Probe on Labelled-Support 6

The oligonucleotide 5′FAM-CCTGCAGGCCCGTGCCCGT 3′ is synthesized on theABI 394 DNA/RNA synthesizer at 0.2 μmole scale with FAM-labelled,polystyrene support 6, (FIG. 2, L=6-carboxyfluorescein FAM,S=polystyrene) The standard 0.2 μmol CE cycle is modified by increasingthe coupling time from 25 s to 90 s for coupling of all5′-phosphoramidite, 3′-DMT phosphoramidite nucleosides (Glen Research).After synthesis in the 5′ to 3′ direction is complete, the amino-linkerphosphoramidite reagent 8 (Glen Research) is coupled as the finalmonomer at the 3′ terminus and detritylated with 3% trichloroacetic acidin CH₂Cl₂. The synthesis column, bearing the 3′-amino, 5′-FAMoligonucleotide on solid-support, is removed from the synthesizer. Twoluer-tipped 1 ml syringes are mounted on each end of the synthesiscolumn. One syringe is filled with 10.7 mg (25 μmole) of TAMRA-CO₂H in500 μl dry dimethylformamide (DMF). The second syringe is filled with9.25 mg (25 μmole) HBTU and 9 μl (50 μmole) diisopropylethylamine in 250μl of 1:1, DMF:CH₃CN. The coupling reagents in the syringes are passedthrough the column by sequentially depressing each plunger. Afterthorough mixing for about one minute, the assembly is left to stand forabout 15 minutes for the coupling reaction to proceed. The reagents arewithdrawn into one syringe and discarded. The synthesis column is washedwith 5 ml 1:1, DMF:CH₃CN, 5 ml CH₃CN, and treated with 1 mlMeOH:t-BuNH₂:H₂O (1:1:2) to cleave 5′-FAM, 3′-N-TAMRA Taqmanself-quenching probe: SEQ. ID. NO. 8 5′ FAM-CCTGCAGGCCCGTGCCCGT-N-TAMRA3′from the support. The supernatant containing the probe is heated anddeprotected at 65° C. for 3 h to remove all protecting groups. The probeis analyzed by reverse-phase HPLC and MALDI-TOF mass spectroscopy whichconfirmed homogeneous purity and identity.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the molecular biology and chemistry artswill clearly understand that many modifications are possible in thepreferred embodiment without departing from the teachings thereof. Allsuch modifications are intended to be encompassed within the followingclaims.

1. A method for synthesis of labelled oligonucleotides comprising the steps of: a. providing a labelled solid-support having the structure

where, S is a solid-support; A is a cleavable linker; X is a moiety with three or more attachment sites; L is a label; Y is selected from the group consisting of oxygen, NH, NR where R is methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl, and sulfur; and P₁ is an acid-cleavable protecting group; b. reacting the labelled solid-support with acid to remove the acid-cleavable protecting group, P₁; and c. adding a 5′-phosphoramidite, 3′ protected nucleoside and an activator, thereby forming a bond between Y and the nucleoside.
 2. The method of claim 1 further comprising the steps of d. capping any unreacted sites on the solid-support; and e. adding an oxidizing reagent.
 3. The method of claim 1 further comprising the steps of d. capping any unreacted sites on the solid-support; e. adding an oxidizing reagent; and f. repeating steps b. to e. until the labelled oligonucleotide is completely synthesized.
 4. The method of claim 3 further comprising the step of deprotecting the labelled oligonucleotide.
 5. The method of claim 3 further comprising the steps of cleaving the labelled compound from the solid-support and deprotecting the labelled oligonucleotide.
 6. The method of claim 1 wherein the solid-support S is selected from the group consisting of polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such.
 7. The method of claim 1 wherein the form of the solid support S is selected from the group consisting of small particles, beads, membranes, frits, slides, plates, micromachined chips, alkanethiol-gold layers, non-porous surfaces, addressable arrays, and polynucleotide-immobilizing media.
 8. The method of claim 1 wherein the cleavable linker A is selected from the group consisting of:


9. The method of claim 1 wherein the linker X is selected from the group consisting of:

where n is 1 to
 12. 10. The method of claim 1 wherein the label L is selected from the group consisting of hybridization-stabilizing moieties, fluorescent dyes, fluorescence quenchers, energy-transfer dye sets, chemiluminescent dyes, amino acids, proteins, peptides, enzymes, and affinity ligands.
 11. The method of claim 10 where the fluorescent dyes and fluorescence quenchers are selected from the group consisting of FAM, TET, HEX, JOE, TAMRA, d-TAMRA, JODA, ROX, VIC, NED, dJON, dR139, DABCYL, DABSYL, malachite green, 4,7-dichloro-fluoresceins, 4,7-dichloro-rhodamines, NTB, and cyanines.
 12. The method of claim 10 where the hybridization-stabilizing moieties are selected from the group consisting of Hoechst 33258, CDPI₁₋₃, MGB1, netropsin, and distamycin.
 13. The method of claim 1 wherein the acid-cleavable protecting group P₁ is selected from the group consisting of DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.
 14. The method of claim 1 wherein the 5′-phosphoramidite, 3′ protected nucleoside monomer has the structure:

where, R is selected from the group consisting of cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl; R₁ and R₂ are individually selected from the group consisting of isopropyl, morpholino, methyl, ethyl, lower alkyl, cycloalkyl, and aryl; P₂ is an exocyclic nitrogen protecting group selected from the group consisting of benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, dimethylformamidine, dialkylformamidine, and dialkylacetamidine; and P₃ is an acid-labile protecting group selected from the group consisting of DMT, MMT, pixyl, trityl, and trialkylsilyl.
 15. The method of claim 3 wherein the oligonucleotide comprises one or more DNA, RNA, and nucleic acid analog monomer units.
 16. The method of claim 15 wherein the nucleic acid analog is selected from the group consisting of nucleobase analogs, sugar analogs, and internucleotide analogs.
 17. The method of claim 16 wherein nucleobase analogs are selected from the group consisting of C-5-alkyl pyrimidine, 2,6-diaminopurine, 2-thiopyrimidine, C-5-propyne pyrimidine, phenoxazine, 7-deazapurine, isocytidine, pseudo-isocytidine, isoguanosine, hypoxanthine, 8-oxopurine, and universal base.
 18. The method of claim 16 wherein sugar analogs are selected from the group consisting of 2′-O-alkyl-ribonucleotides, 2′-O-methyl-ribonucleotides, 2′-O-allyl-ribonucleotides, 2′-allyl ribonucleotides, 2′-halo-ribonucleotides, 2′-O-methoxyethyl-ribonucleotides, 2′-branching group-ribonucleotides, 2′-O-branching group-ribonucleotides, 4′-α-anomeric nucleotides, and 1′-α-anomeric nucleotides.
 19. The method of claim 16 wherein internucleotide analogs are selected from the group consisting of 2-aminoethylglycine (PNA), 2′-5′-linkage, inverted 3′-3′ linkage, inverted 5′-5′ linkage, phosphorothioate, methyl phosphonate, non-bridging N-substituted phosphoramidate, alkylated phosphotriester branched structure, and 3′-N-phosphoramidate.
 20. The method of claim 1 wherein the labelled solid-support comprises a compound of the formula

where S is polystyrene or controlled-pore-glass; and L is a label.
 21. A solid-support comprising a compound of the formula

where, S is a solid-support; A is a cleavable linker; X is a moiety with three or more attachment sites; L is a label; Y is selected from the group consisting of O, NH, NR, and S; R is selected from the group consisting of cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl; P₂ is an exocyclic nitrogen protecting group selected from the group consisting of benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, dimethylformamidine, dialkylformamidine, and dialkylacetamidine; and P₃ is acid-labile protecting group selected from the group consisting of DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.
 22. A solid-support comprising a compound of the formula

where, S is a solid-support; A is a cleavable linker; X is a moiety with three or more attachment sites; L is a label; Y is selected from the group consisting of O, NH, NR, and S; R is cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl; P₂ is an exocyclic nitrogen protecting group selected from the group consisting of benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, dimethylformamidine, dialkylformamidine, and dialkylacetamidine.
 23. A method to synthesize a labelled oligonucleotide comprising: coupling a label reagent with functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid; an oligonucleotide on solid support with nucleophilic functionality consisting of alcohol, amine, or thiol; and a coupling reagent; wherein an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed.
 24. The method of claim 23 wherein the label reagents are selected from the group consisting of hybridization-stabilizing moieties, fluorescent dyes, fluorescence quenchers, energy-transfer dye sets, chemiluminescent dyes, amino acids, proteins, peptides, enzymes, and affinity ligands.
 25. The method of claim 24 where the fluorescent dyes and fluorescence quenchers are selected from the group consisting of FAM, TET, HEX, JOE, TAMRA, d-TAMRA, JODA, ROX, VIC, NED, dJON, dR139, DABCYL, DABSYL, malachite green, 4,7-dichloro-fluoresceins, 4,7-dichloro-rhodamines, NTB, and cyanines.
 26. The method of claim 24 where the hybridization-stabilizing moieties are selected from the group consisting of Hoechst 33258, CDPI₁₋₃, MGB1, netropsin, and distamycin.
 27. The method of claim 23 wherein the labels are attached to the oligonucleotide at label sites consisting of the 5′ terminus, the 3′ terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.
 28. The method of claim 23 wherein the solid-support comprises a compound of the formula

where, S is a solid-support; A is a cleavable linker; X is a moiety with three or more attachment sites; L is a label; Y is selected from the group consisting of oxygen, NH, NR, where R is methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl, and sulfur; and P₁ is an acid-cleavable protecting group.
 29. The method of claim 23 wherein the solid-support is selected from the group consisting of polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic beads, polyacrylate, hydroxethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers or grafts of such.
 30. The method of claim 29 wherein the form of the solid support is selected from the group consisting of small particles, beads, membranes, frits, non-porous surfaces, slides, plates, micromachined chips, alkanethiol-gold layers, addressable arrays, or polynucleotide-immobilizing media.
 31. The method of claim 23 wherein the oligonucleotide comprises one or more DNA, RNA, PNA and nucleic acid analog monomer units.
 32. The method of claim 31 wherein the nucleic acid analog is selected from the group consisting of nucleobase analogs, sugar analogs, and internucleotide analogs.
 33. The method of claim 32 wherein nucleobase analogs are selected from the group consisting of C-5-alkyl pyrimidine, 2,6-diaminopurine, 2-thiopyrimidine, C-5-propyne pyrimidine, phenoxazine, 7-deazapurine, isocytidine, pseudo-isocytidine, isoguanosine, hypoxanthine, 8-oxopurine, and universal base.
 34. The method of claim 32 wherein sugar analogs are selected from the group consisting of 2′-O-alkyl-ribonucleotides, 2′-O-methyl-ribonucleotides, 2′-O-allyl-ribonucleotides, 2′-allyl ribonucleotides, 2′-halo-ribonucleotides, 2′-O-methoxyethyl-ribonucleotides, 2′-branching group-ribonucleotides, 2′-O-branching group-ribonucleotides, 4′-α-anomeric nucleotides, and 1′-α-anomeric nucleotides.
 35. The method of claim 32 wherein internucleotide analogs are selected from the group consisting of 2-aminoethylglycine (PNA), 2′-5′-linkage, inverted 3′-3′ linkage, inverted 5′-5′ linkage, phosphorothioate, methyl phosphonate, non-bridging N-substituted phosphoramidate, alkylated phosphotriester branched structure, and 3′-N-phosphoramidate.
 36. The method of claim 23 wherein the direction of oligonucleotide synthesis is 5′ to 3′.
 35. The method of claim 23 wherein the direction of oligonucleotide synthesis is 3′ to 5′. 